Article Figures & Data

Figures

Gene silencing of Fli1 leads to the induction of SSc-like protein and gene expression profiles in keratinocytes. (A) Immunohistochemistry for Fli1 on the skin sections of healthy controls (n = 8), lcSSc patients with anticentromere antibody (n = 8), and dcSSc patients with anti–topoisomerase I antibody (n = 5). Representative images are shown. Bars, 100 µm. (B) The results of Fli1 staining intensity in A were semiquantitatively evaluated with a four-point grading scale. Kruskal-Wallis test followed by Dunn’s posthoc test was used. (C) mRNA was isolated from skin epidermal sheets from healthy controls (n = 4) and dcSSc patients (n = 6), and gene expressions were analyzed by qRT-PCR. (D) Whole cell lysates from cultured NHKs treated with control nonsilencing SCR or FLI1 siRNA (siFLI1) were subjected to immunoblotting. The values below each blot represent the relative levels of target molecules normalized by controls with densitometry. Representative images of the blots of four independent experiments are shown. (Right) The bar graphs summarize the relative values of the density from these four experiments. (E) The mRNA expression of FLI1, SNAI1, and CDH1 in NHKs treated with SCR or siFLI1 was evaluated. Relative expression levels from four independent experiments were normalized to the expression levels treated with SCR. (F) ChIP assay in NHKs was performed with anti-Fli1 antibody and the primers specific for the designated area of the SNAI1 gene promoter. (G) The back skin taken from Fli1+/− mice and their littermate wild-type (Fli1+/+) mice were immunohistochemically stained for K6 and K16. n = 5 for each group. (Left) Representative images of immunohistochemistry are shown. Bars, 100 µm. (Right) The graphs show the results of semiquantitative scoring of the staining intensity. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; by two-tailed Mann-Whitney U test, unless otherwise indicated. The results are from representative experiments that have been repeated twice independently or are otherwise indicated. AU, arbitrary units; Ctrl, control.

K14Cre;fl/fl mice exhibit SSc-like epidermal phenotypes. (A, left) A representative image of mice at ∼4 mo of age. (Right) The comparison of body weight between two strains of mice. n = 7 per genotype. (B, left) Immunohistochemistry for Fli1 in the back skin of mice. Bars, 50 µm. (Right) The graph summarizes the result of semiquantitative scoring of the staining intensity. n = 7 per genotype. (C) Immunohistochemistry for K6, K16, IL-1α, CTGF, and SNAI1 in the back skin of mice. n = 5 per genotype. Bars, 50 µm. (D) The results of staining intensity in the epidermis as shown in C were evaluated with semiquantitative scoring. n = 5 per genotype. (E) mRNA expressions of these key molecules were determined by qRT-PCR in the epidermal sheets prepared from the back skin of mice. n = 5 per genotype. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; by two-tailed Mann-Whitney U test. The results are from representative experiments that have been repeated twice in different pairs of mice with similar results. Representative images of immunohistochemistry are shown. AU, arbitrary units.

K14Cre;fl/fl mice spontaneously develop SSc-like dermal fibrosis. (A) H&E and Masson-Trichrome staining of the back skin of 3-mo-old mice. Double-headed arrows represent dermal thickness. Bars, 200 µm. (B) The dermal thickness was evaluated at the ages of 1, 2, and 3 mo in each group of mice. (C) Hydroxyproline contents of the back skin were compared between two strains of mice at the ages of 1, 2, and 3 mo. The values are normalized to the value of 1-mo-old fl/fl mice. (D) mRNA expressions of the Col1a1 and Col1a2 genes were measured by qRT-PCR in the back skin of 3-mo-old mice. (E) Skin sections from 3-mo-old mice were stained for α-SMA. (Left) Representative images of the staining, with arrowheads pointing to α-SMA–positive myofibroblasts. Bars, 50 µm. (Right) The bar graph shows the number of α-SMA–positive myofibroblasts per HPF. (F) Adipose tissue thickness in each group of mice was evaluated at the age of 3 mo. (G) Transverse and longitudinal sections of collagen fiber alignment in the superficial dermis were examined by transmission electron microscopy (TEM). (Middle) Higher magnification images of the regions indicated by squares in the top panels. Bars: (top) 1 µm; (middle) 100 nm. (Bottom) Median, range, and frequency distribution profiles obtained by manually measuring the smallest diameter of 300 collagen fibrils are summarized. Colored bars in the histogram indicate the median of each group. n = 3 mice per genotype. (H) mRNA expressions of genes related to collagen fibrillogenesis were determined by qRT-PCR in the back skin of 3-mo-old mice. (I and J) Inflammatory cell infiltrations in the back skin at the ages of 1, 2, and 3 mo were assessed per HPF. Immunostaining for CD3 (I) and F4/80 (J, left) and toluidine blue staining (J, right) were performed to count T cells, macrophages, and mast cells, respectively. (K) mRNA levels of key cytokines, chemokines, and growth factors were assessed by qRT-PCR in the back skin of 3-mo-old mice. Data are shown as mean ± SEM. *, P < 0.05 by two-tailed Mann-Whitney U test. n = 4 – 5 mice per genotype, unless otherwise indicated. The results are from representative experiments that have been repeated three times in different pairs of mice with similar results. Representative images of H&E, Masson-Trichrome, and α-SMA staining and transmission electron microscopy are shown. AU, arbitrary units.

K14Cre;fl/fl mice develop esophageal fibrosis and ILD. (A) Masson-Trichrome staining of the lower esophagus of 3-mo-old fl/fl and K14Cre; fl/fl mice. (Bottom) Higher magnification images of the regions indicated by squares in the top panels. Bars: (top) 500 µm; (bottom) 200 µm. (B) The lower esophagi were cut along the longitudinal direction, spread on sheets, fixed in formalin, and embedded in the paraffin. Masson-Trichrome staining was performed, and the thickness of the lamina propria was evaluated at the ages of 1, 2, and 3 mo. Vertical bars represent lamina propria thickness. Bars, 50 µm. (C) The thickness of lamina propria at upper (thoracic) esophagus was evaluated at the age of 3 mo. (D) The thickness of the circular muscle layer was evaluated in the sections of 3-mo-old mice. (E) Hydroxyproline contents of the lower esophagus were compared between these two strains of mice at the age of 3 mo. (F) mRNA levels of type I collagen, cytokines, chemokines, and growth factors in the lower esophagi of 3-mo-old mice were assessed. (G) The lower esophagus sections from 3-mo-old mice were immunohistochemically stained for IL-1β. Bars, 50 µm. (Right) The graph shows the result of semiquantitative scoring of the staining intensity. (H) H&E staining of the lungs from 1-, 2-, and 3-mo-old fl/fl and K14Cre;fl/fl mice. Arrows indicate the peribronchial lymphocyte aggregates. Bars, 200 µm. (I) Infiltrating inflammatory cells were immunohistochemically stained for CD3 and B220 in the lungs of 3-mo-old K14Cre;fl/fl mice. Bars, 200 µm. (J, left) The Masson-Trichrome staining of the lungs from 3-mo-old fl/fl and K14Cre;fl/fl mice. Bars, 200 µm. (Right) Collagen contents of the total left lung from these mice were measured by hydroxyproline assay. (K) mRNA levels of cytokines, chemokines, and growth factors in the lung of 3-mo-old mice were assessed by qRT-PCR. Data are shown as mean ± SEM. *, P < 0.05 by two-tailed Mann-Whitney U test. n = 4–5 mice per each genotype in all experiments. The results are from representative experiments that have been repeated three times in different pairs of mice with similar results. Representative images of H&E and Masson-Trichrome staining and immunohistochemistry are shown. AU, arbitrary units.

K14Cre;fl/fl mice exhibit prominent autoimmunity with Th2/Th17 cell polarization and abnormal B cell activation leading to autoantibody production. (A) A representative image of the gross appearance of spleens and inguinal lymph nodes from 3-mo-old fl/fl and K14Cre;fl/fl mice. (B) The number of splenocytes in 3-mo-old fl/fl and K14Cre;fl/fl mice was counted. n = 4 per each group. (C) Lymphocytes isolated from inguinal lymph nodes were stained for CD3 and CD4 and intracellularly stained for IL-4, IL-17A, and IFN-γ. (Top) Representative two-dimensional plots for IL-4 and IL-17A in CD3+CD4+ cells. (Bottom) The percentages of cells positive for IL-4, IL-17A, and IFN-γ are summarized. n = 7 mice per each group. (D) Splenocytes from 3-mo-old fl/fl and K14Cre;fl/fl mice were stained for B220 and CD19. B220+CD19+ cells were analyzed for CD19 expression. A representative histogram of mean fluorescence intensity (MFI; top) and the comparison of mean fluorescence intensities (bottom) are shown. n = 4 mice per genotype. (E) Sera from 3- and 8-mo-old mice were analyzed for the concentrations of total IgG, IgM, and IgA. n = 5–7 mice per genotype. Note that the results are shown with log scale. (F) Sera from 1-, 2-, 3-, and 8-mo-old mice were analyzed for the concentration of IL-6. n = 6–7 mice per genotype. (G) B cells were isolated from splenocytes of 3-mo-old mice and cultured for 48 h, either unstimulated or stimulated with anti-CD40 antibody (αCD40Ab). The culture supernatants were subject to the measurement of IL-6 concentration. n = 4 mice per each group. (H) The presence of ANA in the sera was assessed by indirect immunofluorescence with Hep-2 cells. (Left) The photographs show representative immunofluorescence images with sera from 2-mo-old mice. n = 8 mice per genotype. Bars, 100 µm. (Right) Titers of ANA in the sera were assessed with a specific ELISA. The dashed line indicates the mean + 2 SD value of fl/fl mice. n = 4–5 mice per genotype. (I) Immunoblotting of lung tissue lysates from Rag1−/− mice with sera from fl/fl and K14Cre;fl/fl mice. n = 3 mice sera per group. For photographs, immunofluorescence, and immunoblotting, representative results are shown. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; by two-tailed Mann-Whitney U test. The results are from representative experiments that have been repeated three times in different pairs of mice. AU, arbitrary units; MFI, mean fluorescence intensity.

mTECs robustly express Fli1, and K14Cre;fl/fl mice show thymic hypotrophy with decreased Aire expression. (A and B) Immunohistochemistry for Fli1 (A) and double immunofluorescence for Aire and Fli1 (B) were performed with thymuses of 1-mo-old wild-type (C57BL/6) mice. In immunofluorescence, Aire, Fli1, and nuclei were stained with fluorescein isothiocyanate (green), Alexa Flour 555 (red), and DAPI (blue), respectively. Arrowheads indicate Aire-positive mTECs expressing Fli1. n = 5 mice. Bars, 50 µm. (C) Gross appearance of thymuses from 2-mo-old fl/fl and K14Cre;fl/fl mice. (D) The number of thymocytes was counted per lobe. n = 4 mice per genotype. (E) H&E staining of thymuses from 2-mo-old fl/fl and K14Cre;fl/fl mice. c, cortex; m, medulla. (Right) The total area of the medulla and the mean area per islet in each group. n = 4 per each group of mice. Bars, 200 µm. (F) Thymuses from 2-mo-old mice were subjected to flow cytometric analysis. The number of mTECs, defined as CD45−EpCAM+Ly51− cells, was calculated per lobe. n = 4 per group of mice. (G) Immunohistochemistry for Fli1 and double immunofluorescence for Aire and K5 with thymuses from 8-wk-old mice. K5-positive areas indicate the thymic medulla. Bars, 50 µm. (H, top) Fli1- and Aire-positive cells per HPF were counted. (Bottom) The correlation between the numbers of Aire- and Fli1-positive cells was assessed by Spearman’s correlation analysis. The solid line represents the regression line. n = 4 per group. (I) mRNA expression analysis for Fli1, Aire, and representative Aire-dependent and Aire-independent genes in the sorted mTECs defined as CD45−EpCAM+Ly51− cells. n = 4 per group. AU, arbitrary units. (J) Analysis on thymic T reg cells. Gates were set serially on singlets, and CD3+ and CD4+Foxp3+ cells were analyzed. The proportion of CD4+Foxp3+ cells in CD3+ cells (left) and the number of these cells (right) are shown. n = 4 mice per group. (A, E, and G) Dashed lines indicate the border between medulla and cortex. For immunofluorescence, immunohistochemistry, and H&E staining, representative images of five independent experiments are shown. Results of other experiments are from representative experiments that have been repeated three times in different pairs of mice yielding similar results. Data are shown as mean ± SEM. *, P < 0.05 by two-tailed Mann-Whitney U test.

Development of ILD in K14Cre;fl/fl mice is dependent on their autoreactive T cells, whereas skin and esophageal fibrosis is not. (A) Representative H&E and Masson-Trichrome staining of the lungs of recipient Rag1−/− mice to which 106 NK1.1−CD3+ T cells from fl/fl mice or K14Cre;fl/fl mice were transferred 2 (left) and 6 (right) mo before. Bars, 200 µm. (Right) Collagen contents of the total left lung from the recipient mice (2 mo after transfer) were measured by hydroxyproline assay. n = 4–7 mice per group. (B) Immunofluorescence with anti-CD3 antibody and DAPI was performed using the lungs of mice under the same conditions (2 mo after transfer). White, dashed lines indicate the bronchi. Bar, 200 µm. (C) Histological evaluation of inflammatory cell infiltration to various internal organs of recipient Rag1−/− mice 6 mo after transfer. (D) H&E and Masson-Trichrome staining of the back skin of 3-mo-old Rag1−/−;K14Cre;fl/fl and Rag1−/−;fl/fl mice. Double-headed arrows represent dermal thickness. Bars, 200 µm. (E, left) Dermal thickness was evaluated at the age of 3 mo in each group of mice. (Right) Hydroxyproline contents of the back skin were compared between these two groups of mice. (F, top) Masson-Trichrome staining of the lower esophagus of 3-mo-old Rag1−/−;K14Cre;fl/fl and Rag1−/−;fl/fl mice. Bars, 500 µm. (Bottom) Higher magnification images of the regions indicated by squares in the top panels. Bars, 200 µm. (G, left) Lamina propria thickness was evaluated at the age of 3 mo in each group of mice. (Right) Hydroxyproline contents of the lower esophagus were compared between these two groups of mice. n = 4 mice per group, unless otherwise indicated. Data in graphs are shown as mean ± SEM. *, P < 0.05 by two-tailed Mann-Whitney U test. For histological analyses, representative images of three independent experiments are shown. Results of other experiments are from representative experiments that have been repeated twice in different pairs of mice yielding similar results. AU, arbitrary units.

Fli1 directly regulates transcription of the AIRE gene. (A) Luciferase activities were measured in NHKs transfected with the AIRE promoter luciferase construct and either Fli1 expression vector or empty vector (pSG5). n = 4 per group. (B) ChIP in NHKs was performed with anti-Fli1 antibody and the primers specific for the designated areas of the AIRE gene promoter, covering all the EBSs between −1235 and 1. (C) Three putative Fli1 binding sites were mutated as shown in the illustration. Luciferase (Luc) activities were measured in NHKs transfected with wild-type or mutated promoter constructs together with Fli1 expression vector. Fli1-dependent induction of luciferase activities, which is normalized to luciferase activities induced by pSG5 transfection, is shown for each construct. n = 4 per group. The differences were compared with the results of the wild-type promoter construct. (D) The oligonucleotide pull-down assay was conducted using oligonucleotides with wild-type EBS-A or mutated EBS-A (µEBS-A). (Top) A representative blot of three independent experiments. (Bottom) The band density of each blot was measured by densitometry and summarized in the graph. In all the graphs, data are shown as mean ± SEM. *, P < 0.05 by two-tailed unpaired Student’s t test. The results are from representative experiments that have been repeated twice independently, unless otherwise indicated. AU, arbitrary units.